The high prevalence of obesity in the US is a major public health concern, as overweight and obese individuals are at increased risk for many chronic diseases. Obesity stems from an imbalance between total caloric consumption and total energy expenditure (TEE), therefore accurate measurements of TEE play a pivotal role in understanding and ultimately reversing this epidemic. The gold standard for measuring TEE in free-living individuals is the doubly labeled water (DLW) method; major limitations of the method include high cost and precision of only 7% for individual measurements. In this Small Business Innovative Research (SBIR) project, Los Gatos Research (LGR) will develop, fabricate, and test a novel, laser-based analyzer based on LGR's patented, laser-based Off-Axis Integrated Cavity Output Spectroscopy technology to dramatically improve DLW measurements of TEE by measuring 17O/16O to correct for natural isotopic fluctuations in 18O and 2H. The Triple Isotope Water Analyzer (TIWA) will be capable of real-time, simultaneous, high-throughput (45 samples/day) monitoring of 2H/1H, 18O/16O, and 17O/16O in H2O from human bodily fluids, with a target accuracy and precision of better than 0.6% for 2H/1H, 0.15% for 18O/16O, and 0.2% for 17O/16O at natural isotopic abundances. For enriched body waters that have been labeled with 2H and 18O, the accuracy and precision will be comparable to IRMS and better than 1.0% for 2H/1H and 0.2% for 18O/16O. By measuring 17O/16O, the TIWA will allow quantification of isotopic background fluctuation in 18O/16O and 2H/1H during DLW measurements of TEE, thus eliminating the significant constant background assumption made during DLW experiments, and thereby improving measurement precision and significantly reducing the cost of TEE experiments. Based on the excellent correlation measured during Phase I, we estimate that measurement of the isotopic background fluctuation will result in a 40% increase in the precision of DLW tests of TEE, allowing for smaller study sizes while maintaining the statistical power of the measurements and reducing study costs by an estimated 72%! Additionally, the improved precision introduces the possibility of extending this technique from population studies to the accurate assessment of the TEE of a single subject. During Phase II, LGR will continue to work with Prof. Edward Melanson and Prof. John Speakman to demonstrate and apply an improved instrument for simultaneous isotope measurements using minimally prepared, human urine samples. TEE will be measured using the improved, triple isotope method and the results compared with simultaneous, gold-standard measurement of TEE in a metabolic chamber, standard DLW as measured by IRMS, and standard DLW as measured using laser-based isotope analysis. At the conclusion of this research project, LGR will have demonstrated the use of the TIWA for triple-isotope DLW measurements and empirically determined the improvement in TEE accuracy and reduction in the DLW method cost due to the measurement of background fluctuation using 17O/16O.